Summary: Smallpox virus is a Category A bioterrorism/biowarfare agent. The licensed vaccinia virus-based vaccine (Dryvax) is effective, but produces significant and serious adverse events (AE); one of the most deadly AEs is central nervous system (CNS) disease, with children being at greater risk than adults. Fortunately, new, and, it is hoped, safer smallpox vaccines are in development. As a standard regulatory consideration, pre-clinical neurotoxicity assays are used as in an attempt to predict the risk of damage to the human CNS (neurotoxicity) from live virus vaccines; no such validated neurotoxicity assay is available for vaccinia-based smallpox vaccines. To avoid the expense and validation problems inherent in primate testing, we have developed a prototype smallpox vaccine neurotoxicity assay using rodents. Preliminary data indicate this assay can discriminate differences in vaccinia virus strain-specific neurotoxicity among smallpox vaccines (Dryvax, Lister), and laboratory strains (WR, MVA), with WR>Lister>Dryvax>MVA in order of decreasing neuro-toxicity. Here we propose to 1) complete development and validation of the in vivo mouse neurotoxicity assay as a standardized regulatory safety test to expedite the licensing of safer smallpox vaccines, and 2) use this assay as a disease model to study the molecular pathogenesis of vaccinia-based smallpox vaccine neurotoxicity by identifying critical virus-neural cell gene/gene-product interactions. These studies will improve smallpox vaccine safety tests, e.g., small animal, in vitro and molecular biological-based neurotoxicity assays, and can promote new vaccine development, e.g., rational attenuation of smallpox vaccines via targeted mutations.